The following article originally appeared on September 27, 2022 in Biocompare.
Top Tips for In-House Media Preparation
Although liquid media are widely used for culturing cells and running fed-batch processes, they may not always be available for every cell type and application. In these situations, preparing media in-house from powdered stocks is often preferred—an approach that offers many advantages. Here, we explain what’s involved during in-house media preparation and share some suggested best practices.
What are the advantages of preparing media in-house?
There are several reasons for researchers to consider preparing media in-house. With global supply chain issues extending delivery times, preparing media from powdered stocks can mean experiments get completed faster. In addition, the initial purchase price per liter is often lower with powders, although preparation and filtration costs must still be factored in. Another important benefit of in-house preparation is that it allows for the base medium to be made without supplements that may be included in the liquid version of the product. Finally, a do-it-yourself approach can help circumvent the short shelf lives of many liquid products, as well as free up storage space in fridges and freezers.
What is involved in preparing media from powdered stocks?
Preparing media from powdered stocks is relatively straightforward. First, the powder is weighed out and added to the diluent—typically, cell culture grade water. The amount of diluent used should be around 70–80% of the final volume, and should be mixing on a stir plate as the powder is added. Depending on the formulation, it may next be necessary to adjust the pH (usually using 1M NaOH or HCl) to get the powder into solution. Following this, any supplements (e.g., buffering agents such as sodium bicarbonate or amino acids such as L-glutamine) can be added before topping up with further diluent to reach the final amount. Once the solution is fully mixed and the final pH has been verified, the media should be passed through a pre-sterilized filter unit (a pore size of 0.22 µm is generally recommended) to remove any bacteria. Critically, this last step should be performed in a biological safety cabinet to maintain sterility.
Best practices for in-house media preparation
To ensure that in-house media preparations share the same high quality as off-the-shelf liquid products, researchers should follow several best practice recommendations. These include purchasing powdered stocks from a trusted supplier—one who is able to provide a detailed certificate of analysis and comprehensive instructions for rehydration—and confirming that any additives are certified as being of cell culture grade.
Being consistent with how the product is handled is equally important. Even seemingly trivial variations between different media preps could impact cellular growth and skew experimental results. pH should always be shown to fall within the manufacturer’s specified range to avoid influencing biological processes such as metabolism and cell signaling. Likewise, the osmolarity (the total solute concentration within a specific volume) should be within pre-defined limits to both ensure that the powder is fully solubilized and homogenous, and prevent cells from swelling or contracting.
How the resultant solution is filtered must also be carefully considered. Once the filtration stage is reached, using correct aseptic technique is essential since filtration represents the final step of the process before the media is added to living cells. Here, confirming the vacuum pressure is appropriate for the chosen filtration unit is critical. Too high, and there is a risk of crushing the bottle or tearing the filter, which could lead to unwanted contamination. Filter units should come with proof of robust QC, including confirmation of low protein binding (to ensure that media components are not lost due to capture on the filter) and verification that the bottles supplied for storage will not release leachables over time that could compromise cellular growth rates.
Lastly, once the media is ready for use, it should be clearly labeled with information such as the name of the base media, the concentration of any additives, and the preparation date. It should then be stored according to the manufacturer’s instructions and, if not used before the end of the stated shelf-life, be properly discarded.
Corning Life Sciences offers a broad range of powdered media formulations, cell culture grade additives, sterile filtration units, and more.